In rheumatoid arthritis (RA) synovial fibroblasts are activated by growth factors and cytokines to proliferate and to express matrix-degrading proteases and pro-inflammatory cytokines. This contributes to cartilage degradation and joint destruction. To analyse the parameters that lead to activation of synovial fibroblasts, we established a stable human synoviocyte line (K4IM) from a healthy donor by immortalization with SV40 T antigen (TAg). Characterizing the phenotype of the immortalized K4IM cells, we found that they maintained CD44, CD54 (intercellular adhesion molecule; ICAM-1) and CD95 (Fas) expression, but lost the expression of CD106 (vascular cell adhesion molecule 1; VCAM-1) and the receptors for interleukin 1 (IL-1) and platelet-derived growth factor (PDGF). We also monitored normal expression kinetics of transcription factor Egr-1 upon activation with tumor necrosis factor alpha (TNF-alpha) or synovial fluid from RA patients. In addition, we showed that HLA-DR expression could still be upregulated by recombinant interferon gamma (rINF-gamma). The immortalized K4IM cell line therefore represents a valuable and unique tool to study mechanisms that induce or maintain synoviocyte activation.